1. Field of the Invention
The present invention relates generally to the field of molecular biology. In particular, it relates to methods and compositions relating to the regulation of gene transcription and polypeptide expression.
2. Description of the Related Art
Programmed cell death or apoptosis is a fundamental mechanism in the development of an organism, and occurs from embryogenesis throughout life (Vaux et al., 1993). Much remains to be learned about the apoptotic process at the molecular level, however the more classical type of programmed cell death is thought to require activation of a set of genes that lead to DNA fragmentation and subsequent apoptotic morphological changes.
A membrane receptor-like protein, Fas, has been reported to be involved in this process. Fas antigen is a membrane-associated polypeptide that consists of 306 amino acids in mouse (Watanabe-Fukunaga et al., 1992a), and sequence analysis of the full length Fas cDNA revealed that the human Fas molecule has 335 amino acids, with a calculated molecular weight of 37,729 kda. (Itoh et al., 1991; Oehm et al., 1992). The human Fas antigen has a signal peptide of 16 amino acid residues at its amino terminus, an extracellular domain of 157 amino acid residues, a hydrophobic transmembrane domain of 17 amino acid residues and a cytoplasmic tail of 146 amino acids at its carboxyl terminus. The extracellular domain can be divided into three cysteine-rich subdomains. The cytoplasmic region contains the domain that is required for initiation of the apoptotic response (Itoh and Nagata, 1993).
Structural homology places Fas in the superfamily including tumor necrosis factor receptors, nerve growth factor receptor and CD40 (Watanabe-Fukunaga et al., 1992a; Itoh et al., 1991; Oehm et al., 1992; Itoh & Nagata, 1993). In the immune system, the occurrence of massive cell death in the thymus is at least partly due to an apoptotic process mediated through signaling by the Fas antigen (Yonehara et al., 1989; Trauth et al., 1989) that is also involved in T cell-mediated cytotoxicity (Rouvier et al., 1993). Abnormally expressed Fas is observed in the lpr mutant mouse strains (Watanabe-Fukunaga et al., 1992b; Adachi et al., 1993; Wu et al., 1993) that display lymphoproliferative disorders (Frizzera et al., 1989; Saito et al., 1992).
Fas gene expression has been found to be expressed in several tissues, including thymus, spleen, ovary and heart, and on a number of cell types, including activated T- and B-lymphocytes (Watanabe-Fukunaga et al., 1992; Itoh et al., 1991). Its expression is up-regulated by interferon-g (Itoh et al., 1991). Abnormal expression of Fas is observed in the lpr mutant mouse strains which display lymphoproliferative disorders and spontaneous autoimmune disease (Watanabe-Fukunaga et al., 1992; Wu et al., 1993; Adachi et al., 1993). Correction of abnormal expression of Fas in Fas-transgenic MRL-lpr/lpr mice partially abrogates lymphoproliferation and autoantibody production characteristic of this strain (Wu et al., 1994). Recently, the inventors identified an alternatively spliced form of Fas mRNA in human (Cheng et al., 1994). This mRNA encodes a soluble, secreted form of the Fas molecule due to the deletion of an exon encoding the Fas transmembrane domain. Elevated levels of this soluble form of Fas were demonstrated in some patients with SLE. Moreover, administration of soluble Fas to mice at levels comparable to these present in SLE resulted in apparent inhibition of in vivo apoptosis of lymphocytes.
Sequence analysis of the full length human Fas cDNA has revealed that the human Fas molecule has 335 amino acids, with a calculated molecular weight of 37,729 daltons (Itoh et al., 1991; Oehm et al., 1992; Itoh and Nagata, 1993). Structural homology places Fas in the superfamily including tumor necrosis factor (TNF).sup.3 receptors, nerve growth factor (NGF) receptor and CD40 (Watanabe-Fukunaga et al., 1992; Itoh et al., 1991; Oehm et al., 1992; Mallett and Barclay, 1991).
Fas gene has been mapped to the long arm of chromosome 10 in human (Inazawa et al., 1992; Lichter et al., 1992) and the conserved syntatic segment of chromosome 19 in mouse (Watanabe-Fukunaga et al., 1992). However, The gene structure of either human or mouse Fas has not yet been characterized. In this study, we have isolated the human chromosomal DNA for the human Fas apoptotic molecule, determined the exon/intron organization of the gene and characterized the promoter region.
Age-related immune dysfunctions of T cells include thymic involution (Hadden et al., 1992), decreased T cell response to mitogens or antigens (Powers and Belshe, 1993; Flurkey et al., 1992; Kirschmann and Murasko, 1992; Song et al., 1993; McElhaney et al., 1992), altered cytokine expression (Ernst et al., 1993; Ershler,, 1993; al-Rayes et al., 1992; Daynes and Araneo, 1992) and altered phenotype (Okumura et al., 1993; Howard et al., 1992; Thoman et al., 1993). The decrease in spleen or LN T cell responsiveness has been related to an increase in senescent memory T cells (Okumura et al., 1993; Thoman et al., 1993), which exhibit defective phosphorylation after stimulation (Shi and Miller, 1993; Heyeck and Berg, 1993; Patel and Miller, 1992; Witkowski and Miller, 1993; Saini and Sei, 1993). Thymic involution has been proposed to be due either to defects in thymocyte precursors derived from stem cells or a defect in expression of thymic factors or growth factors required for normal T cell development (Hadden et al., 1992; Candore et al., 1992; Goso et al., 1993; Fridkis-Hareli et al., 1992; Li et al., 1992). Recent evidence indicates that depletion of thymocyte stem cells can not completely account for thymic involution with age (Mehr et al., 1993). Evidence to support an age-related deficiency of growth factors has been provided by the observation that replacement of growth factors or hormones can inhibit thymic involution or T cell dysfunction with aging (Hadden et al., 1992; Candore et al., 1992; Goso et al., 1993; Fridkis-Hareli et al., 1992; Li et al., 1992).